Biological synthesis of therapeutic drugs beneficial for human health using microbes offers an alternative production strategy to the methods that are commonly employed such as direct extraction from source organisms or chemical synthesis. In this study, we evaluated the potential for yeast (Saccharomyces cerevisiae) to be used as a catalyst for the synthesis of tranilast and various tranilast analogs (cinnamoyl anthranilates). Several studies have demonstrated that these phenolic amides have antioxidant properties and potential therapeutic benefits including anti-inflammatory, antiproliferative, and antigenotoxic effects. The few cinnamoyl anthranilates naturally produced in plants such as oats and carnations result from the coupling of various hydroxycinnamoyl-CoAs to anthranilic acid.
The worldwide drug market is large and is constantly expanding. Medical drugs used to treat human and animal diseases can be produced chemically or biologically. Even if the biological production is the preferred strategy, it is still rarely used due to the absence of known biosynthetic pathways, the toxicity of intermediate or final products, and poor yields or high recovery costs. Chemically produced drugs usually require large quantities of expensive and non-ecofriendly chemicals. For example, the drug tranilast (FIG. 1a), which belongs to the group of cinnamoyl anthranilate molecules, is manufactured only using organic synthesis methodologies. Tranilast and some of its analogs were recently shown to exhibit antioxidant, antigenotoxic, and antifibrotic activities (Fagerlund et al. 2009; Lee-Manion et al. 2009; Zammit et al. 2009). This synthetic drug (Rizaban, Kissei Pharmaceutical Co, Japan) is currently used in Japan and South Korea as an antihistamine to treat bronchial asthma, atopic dermatitis, allergic conjunctivitis, allergic rhinitis and other allergic disorders (Azuma et al. 1976; Okuda et al. 1984; Komatsu et al. 1988). Tranilast is also used to treat hypertrophic scars, scleroderma and other skin disease related to excessive fibrosis because it has the capacity to inhibit the release of chemical mediators from mast cells and macrophages, and suppresses collagen deposition (reviewed in Isaji et al. 1998). More recently, tranilast was shown to both inhibit and increase the expression of proinflammatory and anti-inflammatory cytokines, respectively, confirming its role in regulating mast cell and macrophage degranulation (Prud'homme 2007; Pae et al. 2008; Sun et al. 2010). Thus, health beneficial effects of tranilast have been assessed in vivo against the development of several disorders associated with pro-inflammatory leukocyte mediators, fibrogenesis and tumorigenesis including atherosclerosis, restenosis after angioplasty, arthritis, lacrimal gland chronic GVHD, inflammatory bowel disease, multiple sclerosis, adhesions, fibrosis, and tumor angiogenesis, growth and metastasis (Tamai et al. 2002; Platten et al. 2005; Oshitani et al. 2007; Chakrabarti et al. 2009; Cui et al. 2009; Guo et al. 2009; Ogawa et al. 2010; Shiota et al. 2010; Tan et al. 2010). Importantly, several years of clinical use have established that tranilast is well tolerated by most patients at doses of up to 600 mg/day for months (Konneh 1998).
Identification of new genes, biochemical characterization of enzymes, and the combination of enzymes to generate biological pathways are key elements of synthetic biology for the engineering of foreign hosts that are able to biologically synthesize naturally- and non-naturally-occurring drugs. Additionally, high-yield production is usually achieved when biosynthetic pathways are heterologously expressed in microbes that are suitable for fermentor production such as yeast Saccharomyces cerevisiae or Escherichia coli. The expression of plant metabolic pathways in microbial organisms is an attractive strategy for the production of valuable natural products that accumulate at low concentrations, are difficult to extract, or originate from endemic plant species (Horwitz 1994; Trantas et al. 2009). Microbial expression systems have several advantages over chemical synthesis or direct extraction from plant tissue, e.g. reduced requirements for toxic chemicals and natural resources, consistant quality, scalability, simple extraction and potential for higher synthesis efficiency (Chang and Keasling 2006). Advantages of Saccharomyces cerevisiae over other microbial hosts include its food-grade status, the extensive knowledge for large scale production, the availability of genetic tools, and its suitability to express plant genes such as cytochrome P450 enzymes (Trantas et al. 2009; Limem et al. 2008). Remarkable examples of pharmaceutical metabolites produced in recombinant yeast strains expressing plant genes include the precursor of the antimalarial drug artemisinic acid and taxadiene (Ro et al. 2006, Engels et al. 2006), flavonoids, stilbenoids and phenylpropanoids (Vannelli et al. 2007; Limem et al. 2008), vitamin C (Branduardi et al. 2007), hydrocortisone (Szczebara et al. 2003), and serotonin derivates (Park et al. 2008).
Natural cinnamoyl anthranilates are produced by the amide condensation of anthranilate and (hydroxy)-cinnamoyl-CoA derivatives, and most of them were co-purified from oats and carnation plants (Ponchet et al. 1988; Collins 1989).